This invention relates to DNA constructs, replicable expression vectors containing the constructs, attenuated bacteria containing the constructs and vaccines containing the said bacteria.
In recent years, there has emerged a new generation of live oral salmonella vaccines based upon strains of Salmonella which have been attenuated by the introduction of a non-reverting mutation in a gene in the aromatic biosynthetic pathway of the bacterium. Such strains are disclosed, for example, in EP-A-0322237. The aforesaid live oral salmonella vaccines are showing promise as vaccines for salmonellosis in man and animals, and they can also be used effectively as carriers for the delivery of heterologous antigens to the immune system. Combined salmonella vaccines have been used to deliver antigens from viruses, bacteria, and parasites, eliciting secretory, humoral and cell-mediated immune responses to the recombinant antigens. Combined salmonella vaccines show great potential as single dose oral multivaccine delivery systems [C. Hormaeche et al, FEMS Symposium No. 63, Plenum, New York; pp 71-83, 1992].
There are problems to be overcome in the development of combined salmonella vaccines. A major consideration is obtaining a high level of expression of the recombinant antigen in the salmonella vaccine so that it will be sufficient to trigger an immune response. However, unregulated high level expression of foreign antigens can be toxic and affect cell viability [I. Charles and G. Dougan, TIBTECH 8, pp 117-21, 1990], rendering the vaccine ineffective or causing loss of the recombinant DNA. Several possible solutions to this problem have been described, such as expression from plasmids carrying essential genes, xe2x80x9con-offxe2x80x9d promoters or incorporation of the foreign genes into the salmonella chromosome.
An alternative approach to overcoming the aforesaid problem would be to use a promoter which is inducible in vivo, and one such promoter is the E. coli nitrite reductase promoter nirB which is induced under anaerobiosis and has been used in biotechnology for the production of tetanus toxin fragment C (TetC) of Clostridium tetani [M. D. Oxer et al Nucl. Ac. Res., 19, pp 2889-92, 1991]. It has previously been found by the inventors of this application (S. N. Chatfield et al Bio/Technology, Vol. 10, pp 888-92 1992) that an Aro Salmonella harbouring a construct expressing TetC from the nirB promoter (pTETnir15) elicited very high anti-tetanus antibody responses in mice. The article by Chatfield et al was published after the priority date of this application.
However, we have also found that when it was attempted to express the P28 antigen from Schistosoma mansoni alone from nirB, the resulting construct was not immunogenic.
Tetanus toxoid has been extensively used as an adjuvant for chemically coupled guest epitopes [D. A. Herrington et al. Nature, 328, pp 257-9 1987]. The potent immunogenicity of TetC in Salmonella suggested to us that it may be possible to exploit this character to promote the immune response of the guest peptides or proteins. However, fusing two proteins together often leads to an incorrectly folded chimaeric protein which no longer retains the properties of the individual components. For example the B subunit of the Vibrio cholerae (CT-B) and E. coli (LT-B) enterotoxins are powerful mucosal immunogens but genetic fusions to these subunits can alter the structure and properties of the carrier and hence their immunogenicity [see M. Sandkvist et al. J. Bacteriol 169, pp 4570-6, 1987, Clements 1990 and M. Lipscombe et al [Mol. Microbiol 5, pp 1385 1990]. Moreover, many heterologous genes expressed in bacteria are not produced in soluble properly folded, or active forms and tend to accumulate as insoluble aggregates [see C. Schein et al. Bio/Technology 6, pp 291-4, 1988 and R. Halenbeck et al; Bio/Technology 7, pp 710-5, 19893.
It is an object of the invention to overcome the aforementioned problems.
We have now found that efficient expression of recombinant antigens, and in particular fusion proteins, can be achieved in bacteria such as salmonellae, by the use of an inducible promoter such as nirB and by incorporating a flexible hinge region between two antigenic components of the fusion protein. The resulting recombinant antigens have been shown to have good immunogenicity. It has also been found, surprisingly, that enhanced expression of a protein can be obtained when a gene coding for the protein is linked to the gene for tetanus toxin C fragment.
Accordingly, in a first aspect, the present invention provides a DNA construct comprising a promoter sequence operably linked to a DNA sequence encoding first and second proteins linked by a hinge region, characterised in that the promoter sequence is one having activity which is induced in response to a change in the surrounding environment.
In another aspect, the invention provides a DNA construct comprising a promoter sequence operably linked to a DNA sequence encoding linked first and second proteins, wherein the first heterologous protein is an antigenic sequence comprising tetanus toxin fragment C or one or more epitopes thereof.
In a further aspect, the invention provides a replicable expression vector, suitable for use in bacteria, containing a DNA construct as hereinbefore defined. In a another aspect, the invention provides a fusion protein, preferably in substantially pure form, the fusion protein comprising linked (e.g. by a hinge region) first and second proteins, the fusion protein being expressed by a replicable expression vector as hereinbefore defined.
In a further aspect the invention provides a process for the preparation of an attenuated bacterium which comprises transforming an attenuated bacterium with a DNA construct as hereinbefore defined.
The invention also provides a vaccine composition comprising an attenuated bacterium, or a fusion protein, as hereinbefore defined, and a pharmaceutically acceptable carrier.
The first and second proteins are preferably heterologous proteins and in particular can be polypeptide immunogens; for example they may be antigenic sequences derived from a virus, bacterium, fungus, yeast or parasite. In particular, it is preferred that the first said protein is an antigenic sequence comprising tetanus toxin fragment C or epitopes thereof.
The second protein is preferably an antigenic determinant of a pathogenic organism. For example, the antigenic determinant may be an antigenic sequence derived from a virus, bacterium, fungus, yeast or parasite.
Examples of viral antigenic sequences for the first and/or second heterologous proteins are sequences derived from a type of human immuno-deficiency virus (HIV) such as HIV-1 or HIV-2, the CD4 receptor binding site from HIV, for example from HIV-1 or -2., hepatitis A or B virus, human rhinovirus such as type 2 or type 14, Herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus (FMDV), rabies virus, rotavirus, influenza virus, coxsackie virus, human papilloma virus (HPV), for example the type 16 papilloma virus, the E7 protein thereof, and fragments containing the E7 protein or its epitopes; and simian immunodeficiency virus (SIV). Examples of antigens derived from bacteria are those derived from Bordetella pertussis (e.g. P69 protein and filamentous haemagglutinin (FHA) antigens), Vibrio cholerae, Bacillus anthracis, and E. coli antigens such as E. coli heat Labile toxin B subunit (LT-B), E. coli K88 antigens, and enterotoxigenic E. coli antigens. Other examples of antigens include the cell surface antigen CD4, Schistosoma mansoni P28 glutathione S-transferase antigens (P28 antigens) and antigens of flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites, eg. parasites of the genus plasmodium or babesia, for example Plasmodium falciparum, and peptides encoding immunogenic epitopes from the aforementioned antigens.
Particular antigens include the full length Schistosoma mansoni P28, and oligomers (e.g. 2, 4 and 8-mers) of the immunogenic P28 aa 115-131 peptide (which contains both a B and T cell epitope), and human papilloma virus E7 protein, Herpes simplex antigens, foot and mouth disease virus antigens and simian immunodeficiency virus antigens.
The promoter sequence is one having activity which is induced in response to a change in the surrounding environment, and an example of such a promoter sequence is one which has activity which is induced by anaerobic conditions. A particular example of such a promoter sequence is the nirB promoter which has been described, for example in International Patent Application PCT/GB92/00387. The nirB promoter has been isolated from E. coli, where it directs expression of an operon which includes the nitrite reductase gene nirB (Jayaraman et al, J. Mol. Biol. 196, 781-788, 1987), and nirD, nirC, cysG (Peakman et al, Eur. J. Biochem. 191, 315-323, 1990). It is regulated both by nitrite and by changes in the oxygen tension of the environment, becoming active when deprived of oxygen, (Cole, Biochem, Biophys. Acta. 162, 356-368, 1968). Response to anaerobiosis is mediated through the protein FNR, acting as a transcriptional activator, in a mechanism common to many anaerobic respiratory genes. By deletion and mutational analysis the part of the promoter which responds solely to anaerobiosis has been isolated and by comparison with other anaerobically-regulated promoters a consensus FNR-binding site has been identified (Bell et al, Nucl, Acids. Res. 17, 3865-3874, 1989; Jayaraman et al, Nucl, Acids, Res. 17, 135-145, 1989). It has also been shown that the distance between the putative FNR-binding site and the xe2x88x9210 homology region is critical (Bell et al, Molec. Microbiol.4, 1753-1763, 1990). It is therefore preferred to use only that part of the nirB promoter which responds solely to anaerobiosis. As used herein, references to the nirB promoter refer to the promoter itself or a part or derivative thereof which is capable of promoting expression of a coding sequence under anaerobic conditions. The preferred sequence, and which contains the nirB promoter is: AATTCAGGTAAATTTGATGTACATCAAATGGTACCCCTTGCTGAATCGTTAAGG TAGGCGGTAGGGCC (SEQ ID NO: 1)
The hinge region is a region designed to promote the independent folding of both the first and second proteins by providing both spatial and temporal separation between the domains.
The hinge region typically is a sequence encoding a high proportion of proline and/or glycine amino acids. The hinge region may be composed entirely of proline and/or glycine amino acids. The hinge region may comprise one or more glycine-proline dipeptide units.
The hinge region may, for example, contain up to about fifteen amino acids, for example at least 4 and preferably 6-14 amino acids, the number of amino acids being such as to impart flexibility between the first and second proteins.
In one embodiment, the hinge region can correspond substantially to the hinge domain of an antibody immunoglobulin. The hinge regions of IgG antibodies in particular are rich in prolines [T. E. Michaelson et al. J. Biol. Chem. 252, 883-9 1977], which are thought to provide a flexible joint between the antigen binding and tail domains.
Without wishing to be bound by any theory, the prolines are thought to form the rigid part of the hinge as the ring structure characteristic of this amino acid hinders rotation around the peptide bond that connects the proline residue with an adjacent amino acid. This property is thought to prevent proline, and adjacent residues, from adopting the ordered structure of an alpha helix or beta strand. Flexibility is thought to be imparted by glycine, the simplest amino acid, with very limited steric demands. Glycine is thought to function as a flexible elbow in the hinge. Other amino acids may be substituted for glycine, particularly those without bulky side-chains, such as alanine, serine, asparagine and threonine.
In one preferred embodiment, the hinge region is a chain of four or more amino acids defining the sequence
xe2x80x94[X]pxe2x80x94Proxe2x80x94[Y]qxe2x80x94Proxe2x80x94[Z]rxe2x80x94
wherein Pro is proline, X and Y are each glycine, or an amino acid having a non-bulky side chain; Z is any amino acid; p is a positive integer; q is a positive integer of from one to ten; and r is zero or a positive integer greater than zero.
The hinge region can be a discrete region heterologous to both the first and second proteins or can be defined by a carboxy-end portion of the first protein or an amino-end portion of the second protein.
Codons which are infrequently utilised in E. coli [H. Grosjean et al, Gene 18, 199-209, 1982] and Salmonella are selected to encode for the hinge, as such rare codons are thought to cause ribosomal pausing during translation of the messanger RNA and allow for the correct folding of polypeptide domains [I. J. Purvis et al. J. Mol. Biol. 193, 413-7 1987]. In addition, where possible restriction enzymes are chosen for the cloning region which, when translated in the resulting fusion, do not encode for bulky or charged side-groups.
In a most preferred aspect, the present invention provides a DNA molecule comprising the nirB promoter operably linked to a DNA sequence encoding first and second polypeptide immunogens linked by a hinge region, wherein the first polypeptide immunogen comprises tetanus toxin fragment C or epitopes thereof.
In another preferred aspect of the invention, there is provided a replicable expression vector, suitable for use in bacteria, containing the nirB promoter sequence operably linked to a DNA sequence encoding first and second polypeptide immunogens linked by a hinge region, wherein the first polypeptide immunogen comprises tetanus toxin fragment C or epitopes thereof.
It has been found that by providing a DNA sequence encoding tetanus toxin fragment C (TetC) linked via a hinge region to a second sequence encoding an antigen, the expression of the sequence in bacterial cells is enhanced relative to constructs wherein the fragment C and hinge region are absent. For example, the expression level of the full length P28 protein of P S. mansoni when expressed as a fusion to TetC was greater than when the P28 protein was expressed alone from the nirB promoter. The TetC fusions to the full length P28 protein of S. mansoni and its tandem epitopes were all soluble and expressed in both E. coli and S.typhimurium. In addition, the TetC-P28 fusion protein was capable of being affinity purified by a glutathione agarose matrix, suggesting that the P28 had folded correctly to adopt a conformation still capable of binding to its natural substrate.
Stable expression of the first and second heterologous proteins linked by the hinge region can be obtained in vivo. The heterologous proteins can be expressed in an attenuated bacterium which can thus be used as a vaccine.
The attenuated bacterium may be selected from the genera Salmonella, Bordetella, Vibrio, Haemophilus, Neisseria and Yersinia. Alternatively, the attenuated bacterium may be an attenuated strain of enterotoxigenic Escherichia coli. In particular the following species can be mentioned: S. typhixe2x80x94the cause of human typhoid; S. typhimuriumxe2x80x94the cause of salmonellosis in several animal species; S. enteritidisxe2x80x94a cause of food poisoning in humans; S. choleraesuisxe2x80x94a cause of salmonellosis in pigs; Bordetella pertussisxe2x80x94the cause of whooping cough; Haemophilus influenzaexe2x80x94a cause of meningitis; Neisseria gonorrhoeaexe2x80x94the cause of gonorrhoea; and Yersiniaxe2x80x94a cause of food poisoning.
Attenuation of the bacterium may be attributable to a non-reverting mutation in a gene in the aromatic amino acid biosynthetic pathway of the bacterium. There are at least ten genes involved in the synthesis of chorismate, the branch point compound in the aromatic amino acid biosynthetic pathway. Several of these map at widely differing locations on the bacterial genome, for example aroA (5-enolpyruvylshikimate-3-phosphate synthase), aroC (chorismate synthase), aroD (3-dihydroquinate dehydratase) and aroE (shikimate dehydrogenase). A mutation may therefore occur in the aroA, aroC, aroD, or aroE gene.
Preferably, however, an attenuated bacterium harbours a non-reverting mutation in each of two discrete genes in its aromatic amino acid biosynthetic pathway. Such bacteria are disclosed in EP-A-0322237. Double aro mutants which are suitable are aroA aroC, aroA aroD, and aroA aroE. Other bacteria having mutations in other combinations of the aroA, aroC, aroD and aroE genes are however useful. Particularly preferred are Salmonella double aro mutants, for example double aro mutants of S. typhi or S. typhimurium, in particular aroA aroC, aroA aroD and aroA aroE mutants. Alternatively, the attenuated bacterium may harbour a non-reverting mutation in a gene concerned with the regulation of one or more other genes (EP-A-0400958). Preferably the mutation occurs in the ompR gene or another gene involved in regulation. There are a large number of other genes which are concerned with regulation and are known to respond to environmental stimuli (Ronson et al, Cell 49, 579-581).
This type of attenuated bacterium may harbour a second mutation in a second gene. Preferably the second gene is a gene encoding for an enzyme involved in an essential biosynthetic pathway, in particular genes involved in the pre-chrorismate pathway involved in the biosynthesis of aromatic compounds. The second mutation is therefore preferably in the aroA, aroC or aroD gene.
Another type of attenuated bacterium is one in which attenuation is brought about by the presence of a non-reverting mutation in DNA of the bacterium which encodes, or which regulates the expression of DNA encoding, a protein that is produced in response to environmental stress. Such bacteria are disclosed in WO 91/15572. The non-reverting mutation may be a deletion, insertion, inversion or substitution. A deletion mutation may be generated using a transposon.
An attenuated bacterium containing a DNA construct according to the invention can be used as a vaccine. Fusion proteins (preferably in substantially pure form) expressed by the bacteria can also be used. in the preparation of vaccines. For example, a purified TetC-P28 fusion protein has been found to be immunogenic on its own. In a further aspect therefore, the invention provides a vaccine composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an attenuated bacterium or fusion protein as hereinbefore defined.
The vaccine may comprise one or more suitable adjuvants.
The vaccine is advantageously presented in a lyophilised form, for example in a capsular form, for oral administration to a patient. Such capsules may be provided with an enteric coating comprising, for example, Eudragit xe2x80x9cSxe2x80x9d, Eudragit xe2x80x9cLxe2x80x9d, Cellulose acetate, Cellulose acetate phthalate or Hydroxypropylmethyl Cellulose. These capsules may be used as such, or alternatively, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is advantageously effected in buffer at a suitable pH to ensure the viability of the organisms. In order to protect the attenuated bacteria and the vaccine from gastric acidity, a sodium bicarbonate preparation is advantageously administered before each administration of the vaccine. Alternatively, the vaccine may be prepared for parenteral administration, intranasal administration or intramammary administration.
The attenuated bacterium containing the DNA construct of the invention may be used in the prophylactic treatment of a host, particularly a human host but also possibly an animal host. An infection caused by a micro-organism, especially a pathogen, may therefore be prevented by administering an effective dose of an attenuated bacterium according to the invention. The bacterium then expresses a heterologous protein or proteins capable of raising antibody to the micro-organism. The dosage employed will be dependent on various factors including the size and weight of the host, the type of vaccine formulated and the nature of the heterologous protein.
An attenuated bacterium according to the present invention may be prepared by transforming an attenuated bacterium with a DNA construct as hereinbefore defined. Any suitable transformation technique may be employed, such as electroporation. In this way, an attenuated bacterium capable of expressing a protein or proteins heterologous to the bacterium may be obtained. A culture of the attenuated bacterium may be grown under aerobic conditions. A sufficient amount of the bacterium is thus prepared for formulation as a vaccine, with minimal expression of the heterologous protein occurring.
The DNA construct may be a replicable expression vector comprising the nirB promoter operably linked to a DNA sequence encoding the tetanus toxin C fragment or epitopes thereof and the second heterologous protein, linked by a hinge region. The nirB promoter may be inserted in an expression vector, which already incorporates a gene encoding one of the heterologous proteins (e.g. tetanus toxin C fragment), in place of the existing promoter controlling expression of the protein. The hinge region and gene encoding the second heterologous protein (e.g. an antigenic sequence) may then be inserted. The expression vector should, of course, be compatible with the attenuated bacterium into which the vector is to be inserted.
The expression vector is provided with appropriate transcriptional and translational control elements including, besides the nirB promoter, a transcriptional termination site and translational start and stop codons. An appropriate ribosome binding site is provided. The vector typically comprises an origin of replication and, if desired, a selectable marker gene such as an antibiotic resistance gene. The vector may be a plasmid.